U.S. patent application number 17/578743 was filed with the patent office on 2022-08-11 for process for preparing substituted polycyclic pyridone derivative and crystal thereof.
This patent application is currently assigned to Shionogi & Co., Ltd.. The applicant listed for this patent is Shionogi & Co., Ltd.. Invention is credited to Kosuke ANAN, Nobuaki FUKUI, Toshikatsu MAKI, Setsuya SHIBAHARA.
Application Number | 20220251107 17/578743 |
Document ID | / |
Family ID | |
Filed Date | 2022-08-11 |
United States Patent
Application |
20220251107 |
Kind Code |
A1 |
SHIBAHARA; Setsuya ; et
al. |
August 11, 2022 |
PROCESS FOR PREPARING SUBSTITUTED POLYCYCLIC PYRIDONE DERIVATIVE
AND CRYSTAL THEREOF
Abstract
The present invention provides a process for preparing a
compound of the formula (II): ##STR00001## wherein R.sup.2 is
unsubstituted alkyl, characterized by reacting a compound of the
formula (I): ##STR00002## wherein R.sup.1 is hydrogen or a
protecting group other than unsubstituted alkyl, with a compound of
the formula: R.sup.2--OH, wherein R.sup.2 is as defined above, in
the presence of a sodium salt and/or a magnesium salt.
Inventors: |
SHIBAHARA; Setsuya;
(Amagasaki-shi, JP) ; FUKUI; Nobuaki;
(Amagasaki-shi, JP) ; MAKI; Toshikatsu;
(Amagasaki-shi, JP) ; ANAN; Kosuke; (Osaka,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Shionogi & Co., Ltd. |
Osaka |
|
JP |
|
|
Assignee: |
Shionogi & Co., Ltd.
Osaka
JP
|
Appl. No.: |
17/578743 |
Filed: |
January 19, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16310897 |
Dec 18, 2018 |
11261198 |
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PCT/JP2017/022478 |
Jun 19, 2017 |
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17578743 |
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International
Class: |
C07D 498/14 20060101
C07D498/14 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 20, 2016 |
JP |
2016-121453 |
Claims
1. A process for preparing a compound of the formula (II):
##STR00043## or a salt thereof, wherein R.sup.2 is unsubstituted
alkyl; the process comprising reacting a compound of the formula
(I): ##STR00044## wherein R.sup.1 is hydrogen or a protecting group
other than unsubstituted alkyl, with a compound of the formula:
R.sup.2--OH, wherein R.sup.2 is as defined above, in the presence
of a sodium salt and/or a magnesium salt.
2. The process according to claim 1, wherein the reaction is
carried out in the presence of a magnesium salt.
3. The process according to claim 1, wherein the reaction is
carried out in the presence of isopropyl magnesium chloride.
4. The process according to claim 1, wherein R.sup.1 is benzyl.
5. The process according to claim 1, wherein R.sup.2 is hexyl.
6-8. (canceled)
9. Currently Amended A compound of the formula (II'): ##STR00045##
or a salt thereof.
10. A tosylate salt of the compound of formula (II') according to
claim 9.
11. A crystal of the tosylate salt according to claim 10.
12-15. (canceled)
16. The crystal according to claim 11, comprising an X-ray powder
diffraction pattern wherein the diffraction angles (2.theta.) of at
least two peaks are selected from the group consisting of
5.9.+-.0.2.degree., 8.4.+-.0.2.degree., 11.6.+-.0.2.degree.,
12.7.+-.0.2.degree., 13.1.+-.0.2.degree. and
15.7.+-.0.2.degree..
17. The crystal according to claim 11, comprising an X-ray powder
diffraction pattern comprising peaks at the diffraction angles
(2.theta.) of 5.9.+-.0.2.degree., 8.4.+-.0.2.degree.,
11.6.+-.0.2.degree., 12.7.+-.0.2.degree., 13.1.+-.0.2.degree. and
15.7.+-.0.2.degree..
18. The process according to claim 1, wherein the reaction is
carried out in the presence of cyclohexyl magnesium chloride.
19. The process according to claim 1, wherein the reaction is
carried out in the presence of a sodium salt.
20. The process according to claim 1, wherein the reaction is
carried out in the presence of sodium hydroxide.
21. The process according to claim 1, wherein the reaction is
carried out in the presence of sodium hydride.
22. The process according to claim 1, wherein the reaction is
carried out in the presence of sodium isopropoxide.
23. The process according to claim 1, wherein the reaction is
carried out in the presence of sodium tert-pentoxide.
24. The process according to claim 1, which further comprises
reacting the compound of formula (I) with the compound of formula
(II) in a solvent.
25. The process according to claim 24, wherein the solvent is at
least one selected from the group consisting of dichloromethane,
toluene and tetrahydrofuran.
26. A process for preparing a compound of the formula: ##STR00046##
or a salt thereof, the process comprising reacting a compound of
the formula: ##STR00047## with a compound of the formula:
CH.sub.3(CH.sub.2).sub.5OH, in the presence of a sodium salt and/or
a magnesium salt.
27. The process according to claim 26, which further comprises
reacting the compounds in a solvent.
Description
TECHNICAL FIELD
[0001] The present invention relates to a process for preparing
substituted polycyclic pyridone derivatives and crystals thereof.
Specifically, the present invention relates a process for preparing
substituted polycyclic pyridone derivatives having cap-dependent
endonuclease inhibitory activity and intermediates thereof.
BACKGROUND ART
[0002] WO2010/110409 (Patent Document 1) discloses a process for
preparing a polycyclic pyridone derivative using a pyrone
derivative and a pyridone derivative (Example 3).
##STR00003##
[0003] WO2010/147068 (Patent Document 2) and WO2012/039414 (Patent
Document 3) disclose a process using a pyridone derivative for the
preparation of a polycyclic pyridone derivative (Example 165).
##STR00004## ##STR00005##
[0004] However, Patent Documents 1 to 3 do not describe that use of
a benzyl-protected polycyclic pyridone derivative in a coupling
step of an optically active polycyclic pyridone derivative with a
thiepin derivative reduce the optical purity of product. Also,
there is neither description nor suggestion in the Patent Documents
1 to 3 that the coupling reaction proceeds in good yield without
reduction in optical purity when the coupling reaction is carried
out using a hexyl-protected polycyclic pyridone derivative.
Furthermore, there is neither description nor suggestion that the
reaction proceeds in high yield without reduction in optical purity
when the reaction is carried out in the presence of a magnesium
salt in the reaction to exchange the protecting group in the
polycyclic pyridone derivative from a protecting group other than
unsubstituted alkyl to unsubstituted alkyl.
[0005] Patent Document 1 discloses the following process comprising
a step of coupling a benzyl-protected polycyclic pyridone
derivative with a benzhydryl derivative (Example 21). However,
there is neither description nor suggestion of any step to exchange
the protective group in the polycyclic pyridone derivative.
##STR00006##
[0006] Patent Document 2 discloses a step of coupling a substituted
tricyclic pyridone derivative with a benzhydryl derivative (Example
175). However, there is neither description nor suggestion of any
step to exchange the protective group in the tricyclic pyridone
derivative.
##STR00007## ##STR00008##
[0007] Patent Document 2 discloses a step of coupling a substituted
tricyclic pyridone derivative with a thiepin derivative (Examples
583 and 584). However, there is neither description nor suggestion
of any step to exchange the protective group the tricyclic pyridone
derivative or reduction of the optical purity.
##STR00009##
PRIOR ART DOCUMENTS
Patent Documents
[0008] WO2010/110409 [0009] WO2010/147068 [0010] WO2012/039414
SUMMARY
Technical Problem
[0011] PCT/JP2016/63139 describes that the compound, which is the
compound of formula (V) or (VI) as disclosed herein, has a
cap-dependent endonuclease inhibitory activity and is useful as a
therapeutic and/or prophylactic agent for symptoms and/or diseases
caused by infection of influenza virus.
[0012] One object of the present invention is to provide a novel
and useful process for the preparation of substituted polycyclic
pyridone derivatives of formula (V) or (VI) having cap-dependent
endonuclease inhibitory activity and intermediates thereof of
formula (II) or (IV).
Solution to Problem
[0013] The present inventors have found a reduction in the optical
purity of an optically active substituted cyclic pyridone
derivative occurs in a coupling step of an optically active
substituted tricyclic pyridone derivative with a thiepin
derivative.
[0014] The present inventors also have found a process to achieve a
coupling reaction of an optically active substituted tricyclic
pyridone derivative with a thiepin derivative without causing a
reduction of the optical purity, by exchanging from a protective
group other than unsubstituted alkyl, such as benzyl group, to
hexyl group.
[0015] The present invention relates to the following.
(1) A process for preparing a compound of the formula (II):
##STR00010##
wherein R.sup.2 is unsubstituted alkyl, characterized by reacting a
compound of the formula (I):
##STR00011##
wherein R.sup.1 is hydrogen or a protecting group other than
unsubstituted alkyl, with a compound of the formula: R.sup.2--OH,
wherein R.sup.2 is as defined above, in the presence of a sodium
salt and/or a magnesium salt. (2) The process according to (1)
wherein the reaction is carried out in the presence of a magnesium
salt. (3) The process according to (1), wherein the reaction is
carried out in the presence of isopropyl magnesium chloride. (4)
The process according to any one of (1) to (3) wherein R.sup.1 is
benzyl. (5) The process according to any one of (1) to (4) wherein
R.sup.2 is hexyl. (6) A process for preparing a compound of the
formula (IV):
##STR00012##
wherein R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are each
independently hydrogen or halogen, provided that one or two of
R.sup.3, R.sup.4, R.sup.5 and R.sup.6 is halogen, characterized by
reacting a compound of the formula (II'):
##STR00013##
with a compound of the formula (III):
##STR00014##
wherein R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are as defined above.
(7) The process according to (6) wherein R.sup.3 is hydrogen,
R.sup.4 is hydrogen, R.sup.5 is fluorine, and R.sup.6 is fluorine.
(8) A process for preparing the compound of the formula (V) or
formula (VI):
##STR00015##
which comprises the process according to any one of (1) to (7). (9)
A compound of the formula (II'):
##STR00016##
or a salt thereof. (10) The salt of the compound according to (9)
which is a tosylate. (11) A crystal of the salt according to (10).
(12) The crystal according to (11) characterized by an X-ray powder
diffraction pattern wherein the diffraction angles (2.theta.) of at
least two peaks are selected from the group consisting of
5.9.+-.0.2.degree., 8.4.+-.0.2.degree., 11.6.+-.0.2.degree.,
12.7.+-.0.2.degree., 13.1.+-.0.2.degree. and 15.7.+-.0.2.degree..
(13) The crystal according to (11) characterized by an X-ray powder
diffraction pattern comprising peaks at the diffraction angles
(2.theta.) of 5.9.+-.0.2.degree., 8.4.+-.0.2.degree.,
11.6.+-.0.2.degree., 12.7 0.2.degree., 13.1.+-.0.2.degree. and
15.7.+-.0.2.degree.. (14) The crystal according to (11)
characterized by a powder X-ray diffraction spectrum substantially
identical with FIG. 4. (15) A compound of the formula (IV'):
##STR00017##
or a salt thereof. (16) The salt of the compound according to (15)
which is a mesylate. (17) A crystal of the salt according to (16).
(18) The crystal according to (17) characterized by an X-ray powder
diffraction pattern wherein the diffraction angles (2.theta.) of at
least two peaks are selected from the group consisting of
7.1.+-.0.2.degree., 9.3.+-.0.2.degree., 12.6.+-.0.2.degree.,
14.1.+-.0.2.degree., 17.7.+-.0.2.degree., 18.7.+-.0.2.degree.,
19.2.+-.0.2.degree., 22.2.+-.0.2.degree., 25.4.+-.0.2.degree.,
27.7.+-.0.2.degree., 28.5.+-.0.2.degree., and 37.8.+-.0.2.degree..
(19) The crystal according to (17) characterized by an X-ray powder
diffraction pattern comprising peaks at the diffraction angles
(2.theta.) of 7.1.+-.0.2.degree., 9.3.+-.0.2.degree.,
12.6.+-.0.2.degree., 14.1.+-.0.2.degree., 17.7.+-.0.2.degree.,
18.7.+-.0.2.degree., 19.2.+-.0.2.degree., 22.2.+-.0.2.degree.,
25.4.+-.0.2.degree., 27.7.+-.0.2.degree., 28.5.+-.0.2.degree., and
37.8.+-.0.2.degree.. (20) The crystal according to (17) having the
melting point of 219.degree. C..+-.2.degree. C. in differential
scanning calorimetry. (21) The crystal according to (17)
characterized by a powder X-ray diffraction spectrum substantially
identical with FIG. 5. (22) A compound of the formula (VII):
##STR00018##
or a salt thereof. (23) A monohydrate of the compound according to
(22). (24) The monohydrate according to (23) characterized by an
X-ray powder diffraction pattern wherein the diffraction angles
(2.theta.) of at least two peaks are selected from the group
consisting of 5.4.+-.0.2.degree., 7.5.+-.0.2.degree.,
8.4.+-.0.2.degree., 10.6.+-.0.2.degree., 11.9.+-.0.2.degree.,
13.5.+-.0.2.degree., 20.2.+-.0.2.degree. and 22.9.+-.0.2.degree..
(25) The monohydrate according to (23) characterized by an X-ray
powder diffraction pattern comprising peaks at the diffraction
angles (2.theta.) of 5.4.+-.0.2.degree., 7.5.+-.0.2.degree.,
8.4.+-.0.2.degree., 10.6.+-.0.2.degree., 11.9.+-.0.2.degree.,
13.5.+-.0.2.degree., 20.2.+-.0.2.degree. and 22.9.+-.0.2.degree..
(26) The monohydrate according to (23) characterized by a powder
X-ray diffraction spectrum substantially identical with FIG. 1.
(27) A solvate of the compound of the formula (VIII):
##STR00019##
(28) A 1/2 hydrate of the compound of the formula (VIII). (29) The
1/2 hydrate according to (28) characterized by an X-ray powder
diffraction pattern wherein the diffraction angles (2.theta.) of at
least two peaks are selected from the group consisting of
9.5.+-.0.2.degree., 13.4.+-.0.2.degree., 18.0.+-.0.2.degree.,
19.3.+-.0.2.degree., 21.2.+-.0.2.degree., 22.5.+-.0.2.degree.,
22.8.+-.0.2.degree., 23.6.+-.0.2.degree., 27.5.+-.0.2.degree., and
28.1.+-.0.2.degree.. (30) The 1/2 hydrate according to (28)
characterized by an X-ray powder diffraction pattern comprising
peaks at the diffraction angles (2.theta.) of 9.5.+-.0.2.degree.,
13.4.+-.0.2.degree., 18.0.+-.0.2.degree., 19.3.+-.0.2.degree.,
21.2.+-.0.2.degree., 22.5.+-.0.2.degree., 22.8.+-.0.2.degree.,
23.6.+-.0.2.degree., 27.5.+-.0.2.degree., and 28.1.+-.0.2.degree..
(31) The 1/2 hydrate according to (28) characterized by a powder
X-ray diffraction spectrum substantially identical with FIG. 2.
(32) A compound of the formula (IX):
##STR00020##
a salt thereof or a solvate thereof. (33) A crystal of a compound
of the formula (IX). (34) The crystal according to (33)
characterized by an X-ray powder diffraction pattern wherein the
diffraction angles (2.theta.) of at least two peaks are selected
from the group consisting of 7.1.+-.0.2.degree.,
14.1.+-.0.2.degree., 15.1.+-.0.2.degree., 21.0.+-.0.2.degree.,
21.2.+-.0.2.degree., 22.9.+-.0.2.degree., and 23.4.+-.0.2.degree..
(35) The crystal according to (33) characterized by an X-ray powder
diffraction pattern comprising peaks at the diffraction angles
(2.theta.) of 7.1.+-.0.2.degree., 14.1.+-.0.2.degree.,
15.1.+-.0.2.degree., 21.0.+-.0.2.degree., 21.2.+-.0.2.degree.,
22.9.+-.0.2.degree., and 23.4.+-.0.2.degree.. (36) The crystal
according to (33) characterized by a powder X-ray diffraction
spectrum substantially identical with FIG. 3. (37) A crystal of the
compound of the formula (V):
##STR00021##
or a crystal of a pharmaceutically acceptable salt thereof. (38)
The crystal according to (37) characterized by an X-ray powder
diffraction pattern wherein the diffraction angles (2.theta.) of at
least two peaks are selected from the group consisting of
9.6.+-.0.2.degree., 10.9.+-.0.2.degree., 17.8.+-.0.2.degree.,
21.5.+-.0.2.degree., 22.1.+-.0.2.degree., 23.5.+-.0.2.degree., and
24.8.+-.0.2.degree.. (39) The crystal of the compound according to
(37) characterized by an X-ray powder diffraction pattern
comprising peaks at the diffraction angles (2.theta.) of
9.6.+-.0.2.degree., 10.9.+-.0.2.degree., 17.8.+-.0.2.degree.,
21.5.+-.0.2.degree., 22.1.+-.0.2.degree., 23.5.+-.0.2.degree. and
24.8.+-.0.2.degree.. (40) The crystal of the compound according to
(37) characterized by a powder X-ray diffraction spectrum
substantially identical with FIG. 6.
[0016] According to the process of the present invention, a
polycyclic pyridone derivative of the formula (V) or (VI) can be
efficiently prepared with high optical purity.
BRIEF DESCRIPTION OF DRAWINGS
[0017] FIG. 1 is a powder X-ray diffraction pattern of Compound
3.
[0018] FIG. 2 is a powder X-ray diffraction pattern of Compound
9.
[0019] FIG. 3 is a powder X-ray diffraction pattern of Compound
13.
[0020] FIG. 4 is a powder X-ray diffraction pattern of a tosylate
of Compound 20.
[0021] FIG. 5 is a powder X-ray diffraction pattern of a mesylate
of Compound 21.
[0022] FIG. 6 is a powder X-ray diffraction pattern of Compound
(V).
[0023] FIG. 7 is a time-course of the concentration in plasma of
the compound of formula (V) after oral administration of the
compound of formula (VI), which is a prodrug of the compound of
formula (V), to rats under non-fasting condition.
[0024] FIG. 8 is a time-course of the concentration in plasma of
the compound of formula (VI) after oral administration of the
compound of formula (VI), which is a prodrug of the compound of
formula (V), to rats under non-fasting condition.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0025] The meanings of the terms as used herein are explained
below. Unless otherwise specified, each term has the same meaning
when used alone or in combination with other terms.
[0026] The term "consisting of" means to have only the described
elements.
[0027] The term "comprising" means not to limit to the described
elements and not to exclude undescribed elements.
[0028] "Halogen" includes fluorine, chlorine, bromine or iodine.
Fluorine and chlorine are preferable, and fluorine is particularly
preferable.
[0029] "Alkyl" means a C1 to C6 straight or branched alkyl, and
includes C1 to C4 alkyl, C1 to C3 alkyl and the like. Examples
include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,
sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, hexyl,
isohexyl and the like.
[0030] Example of the protecting group other than unsubstituted
alkyl of R.sup.1 includes benzyl.
[0031] Example of the unsubstituted alkyl of R.sup.2 includes
methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,
tert-butyl, n-pentyl, isopentyl, neopentyl, hexyl, and isohexyl;
and n-propyl, isobutyl, hexyl and the like are preferable; and
hexyl is particularly preferable.
[0032] "Protecting group other than unsubstituted alkyl" is not
limited so long as it is a protecting group other than the above
"alkyl" and it is removed in the presence of sodium salt and/or
magnesium salt. Example includes substituted alkyl and the like,
preferably benzyl and the like.
[0033] "Sodium salt" is not limited so long as it is able to remove
"protecting group other than alkyl". Examples include sodium
hydroxide, sodium hydride, sodium isopropyl oxide, sodium
tert-pentoxide, isopropyl magnesium chloride and the like.
Preferred are sodium tert-pentoxide and isopropyl magnesium
chloride, and isopropyl magnesium chloride is particularly
preferable.
[0034] Preferred embodiments of R.sup.1, R.sup.2, R.sup.3, R.sup.4,
R.sup.5, R.sup.6 and "sodium salt and/or magnesium salt" are
described below. Compounds having a possible combination of the
following embodiments are preferable.
[0035] R.sup.1 includes hydrogen or a protecting group other than
unsubstituted alkyl. In a preferred embodiment, R.sup.1 is a
protecting group other than unsubstituted alkyl, and benzyl is
particularly preferable.
[0036] R.sup.2 includes unsubstituted alkyl. In a preferred
embodiment, R.sup.2 includes methyl, ethyl, n-propyl, isopropyl,
n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl,
neopentyl, hexyl, isohexyl and the like, and n-propyl, isobutyl,
hexyl and the like are preferable, and hexyl is particularly
preferable.
[0037] In a preferred embodiment, "sodium salt and/or magnesium
salt" is preferably "magnesium salt", and isopropyl magnesium
chloride, cyclohexyl magnesium chloride and the like are more
preferable, and isopropyl magnesium chloride is particularly
preferable.
[0038] R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are each independently
hydrogen or halogen, and the number of halogen in R.sup.3, R.sup.4,
R.sup.5 and R.sup.6 is one or two.
[0039] In a preferred embodiment, R.sup.3 is hydrogen.
[0040] In a preferred embodiment, R.sup.4 is hydrogen.
[0041] In a preferred embodiment, R.sup.5 is fluorine.
[0042] In a preferred embodiment, R.sup.6 is fluorine.
[0043] The term "the number of halogen in R.sup.3, R.sup.4, R.sup.5
and R.sup.6 is one or two", as used herein, means that one or two
of R.sup.3, R.sup.4, R.sup.5 and R.sup.6 is halogen.
[0044] In the present description, reacting a compound with a
compound includes reacting a salt of such compound or a solvate
thereof.
[0045] Examples of the pharmaceutically acceptable salt of the
compound of the present invention include salts with alkaline
metals (e.g., lithium, sodium, potassium, etc.), alkaline earth
metals (e.g., calcium, barium, etc.), magnesium, transition metals
(e.g., zinc, iron, etc.), ammonia, organic bases (e.g.,
trimethylamine, triethylamine, dicyclohexylamine, ethanolamine,
diethanolamine, triethanolamine, meglumine, ethylenediamine,
pyridine, picoline, quinolin, etc.) or amino acids, or salts with
inorganic acids (e.g., hydrochloric acid, sulfuric acid, nitric
acid, carbonic acid, hydrobromic acid, phosphoric acid, hydroiodic
acid, etc.), or organic acids (e.g., formic acid, acetic acid,
propionic acid, trifluoroacetic acid, citric acid, lactic acid,
tartaric acid, oxalic acid, maleic acid, fumaric acid, mandelic
acid, glutaric acid, malic acid, benzoic acid, phthalic acid,
ascorbic acid, benzenesulfonic acid, p-toluenesulfonic acid,
methanesulfonic acid, ethanesulfonic acid, etc.), particularly,
salts with hydrochloric acid, sulfuric acid, phosphoric acid,
tartaric acid, methanesulfonic acid and the like. These salts can
be formed in accordance with the conventional methods.
[0046] Examples of the pharmaceutically acceptable salt of the
compound of formula (V) include salts with alkaline metals (e.g.,
lithium, sodium, potassium, etc.), alkaline earth metals (e.g.,
calcium, barium, etc.), magnesium, transition metals (e.g., zinc,
iron, etc.), and salts with alkaline metals (e.g., lithium, sodium,
potassium, etc.) and salts with alkaline earth metals (e.g.,
calcium, barium, etc.) are preferable.
[0047] The compound of the present invention or a pharmaceutically
acceptable salt thereof may form a solvate, such as hydrate, and/or
a crystalline polymorph, and the present invention includes such
various solvates as well as crystalline polymorphs. "Solvates" may
be those wherein any numbers of solvent molecules (e.g., water
molecules or the like) are coordinated with the compound of present
the invention. When the compound of the present invention or a
pharmaceutically acceptable salt thereof is allowed to stand in the
atmosphere, it may absorb water, resulting in attachment of
adsorbed water or formation of hydrates. In addition, the compound
of the present invention or a pharmaceutically acceptable salt
thereof may be recrystallized to form a crystal polymorphism.
[0048] A method for characterizing the crystal of the present
invention is illustrated below. Unless otherwise mentioned, the
numerical values in the description and claims are approximate
values. The numerical values may vary due to instrument
calibration, instrument error, material purity, crystal size,
sample size, and other factors.
[0049] The term "crystal" as used herein means a material having an
ordered long-range molecular structure. The degree of crystallinity
of the crystalline form can be measured by a number of techniques
including, for example, powder X-ray diffraction, moisture
adsorption, differential analysis, calorimetric analysis, solution
colorimetry, dissolution properties.
[0050] In general, a crystalline organic compound is composed of a
large number of atoms periodically arranged in a three-dimensional
space. The structural periodicity normally manifests distinct
physical properties that are clearly distinguishable by most
spectroscopic probes (e.g., X-ray diffraction, infrared spectra,
Raman spectra and solid state NMR).
[0051] Among others, X-ray powder diffraction (XRPD) is
acknowledged to be one of the most sensitive methods to determine
the crystallinity of solids. X-rays which are irradiated to
crystals are reflected by the crystal lattice planes and mutually
interfere. Then, only the diffraction lines in the direction which
fulfill the conditions predicted by Bragg's law are intensified,
and the intensity of the order diffraction lines are canceled and
not observed. On the other hand, in the case of amorphous solids,
the ordered diffraction lines over a long-range are not observed.
Amorphous solids usually exhibit a broad XRPD pattern called halo
pattern because of the absence of the long range order of repeating
crystal lattice.
[0052] A crystalline form of the polycyclic pyridone derivatives,
intermediates, salts thereof and/or solvates thereof disclosed in
this description preferably has a distinguishable X-ray powder
diffraction profile. For example, a crystalline form of the
compound of formula (V) can be preferably distinguished by the
presence of characteristic diffraction peaks. The characteristic
diffraction peaks as used herein are those selected from an
observed diffraction pattern. Preferably, the characteristic
diffraction peaks are selected from the diffraction pattern among
approximately twenty peaks, more preferably approximately ten
peaks, and most preferably approximately five peaks.
[0053] In general, it is known that the relative intensities of
various peaks in the Tables and Figures as shown below may vary due
to a number of factors, such as the orientation effects of crystals
on the X-ray beam, the purity of the material to be analyzed or the
degree of crystallinity of the sample. The peak positions may also
shift for variations in the sample height. Furthermore, a
measurement using a different wavelength will result in a different
shift according to the Bragg equation (n.lamda.=2d sin .theta.).
Such XPRD patterns obtained by using a different wavelength are
within the scope of the present invention.
[0054] The crystalline form of the present invention can be
characterized by means of thermal analysis.
DSC (Differential Scanning Calorimetry)
[0055] DSC is one of principal measuring methods for thermal
analysis and a method of measuring the thermal properties of the
substance as an aggregate of atoms/molecules. A differential
scanning calorimetry curve can be obtained by measuring change of
heat capacity over temperature or time of a pharmaceutical active
ingredient by DSC, and plotting the obtained data to temperatures
or times. The information of the onset temperature, endothermic
maximum and enthalpy of melting a pharmaceutical active ingredient
can be obtained from a differential scanning calorimetry curve.
(Preparation of Compound of the Present Invention)
[0056] A general method for preparation of the compound of the
present invention is exemplified below. Further, extraction,
purification and the like may be carried out by conventional
methods practiced in organic chemistry experiments.
[0057] The synthesis of the compound of the present invention can
be carried out with reference to methods known in the art.
[0058] As a raw material compound, commercially available
compounds, compounds described in the present description,
compounds described in the references cited in the present
description, and other known compounds can be utilized.
[0059] If a salt of the compound of the present invention is
desired, it may be purified as it is in the case where the compound
of the present invention is obtained in the form of a salt. In case
where the compound is obtained in a free form, it is dissolved or
suspended in a suitable organic solvent and added with an acid or a
base to form a salt by an ordinary method.
[0060] In addition, the compound of the present invention and a
pharmaceutically acceptable salt thereof may exist in a form of
adduct with water or various solvents (hydrate or solvate). The
present invention also include such adducts.
[0061] The wedge and dotted-lines indicate absolute
configuration.
[0062] The process of the present invention can be carried out, for
example, as follows.
Step 1
##STR00022##
[0063] wherein R.sup.1 is hydrogen or a protecting group other than
unsubstituted alkyl and R.sup.2 is unsubstituted alkyl.
[0064] In this step, a compound of the formula (I) is reacted with
an alcohol of the formula: R.sup.2--OH in the presence of a sodium
salt and/or a magnesium salt to give a compound of the formula
(II).
[0065] The solvent is not limited so long as it allows the above
process to proceed efficiently. Examples of such solvent include
dichloromethane, toluene, tetrahydrofuran and the like, which may
be used alone or in combination. The reaction can be carried out in
a single of mixed solvent, or without solvent. Preferred solvent is
tetrahydrofuran.
[0066] Examples of the sodium salt and/or magnesium salt include
sodium hydroxide, sodium hydride, sodium isopropoxide, sodium
tert-pentoxide, isopropyl magnesium chloride, cyclohexyl magnesium
chloride and the like. Preferred is isopropyl magnesium chloride.
The salt may be used in an amount of 0.1 to 5 molar equivalents,
preferably 0.3 to 0.5 molar equivalents, to the compound (I).
[0067] The reaction temperature is not limited, but the reaction
usually can be conducted at about 0 to 100.degree. C., preferably
at 0.degree. C. to room temperature.
[0068] The reaction time is not limited, but the reaction usually
can be conducted for 0.5 hour to 24 hours, preferably for 1 to 10
hours.
Step 2
##STR00023##
[0069] wherein R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are each
independently hydrogen or halogen provided that one or two of
R.sup.3, R.sup.4, R.sup.5 and R.sup.6 is halogen. Other symbols are
as defined above.
[0070] In this step, a compound of the formula (II') is reacted
with a compound of the formula (III) in the presence of a
condensing agent to obtain a compound of the formula (IV).
[0071] The solvent is not limited so long as it allows the above
step to proceed efficiently. Example of the solvent include ethyl
acetate, cyclohexane, isopropyl acetate, propyl acetate, toluene,
1,4-dioxane, DMA, DMF, toluene, heptane, cyclopentyl methyl ether
and the like, which may be used alone or in combination. The
reaction can be carried out in a single of mixed solvent, or
without solvent. Preferred solvent is a mixed solvent of ethyl
acetate and cyclohexane.
[0072] Examples of the condensing agent include propylphosphonic
anhydride, methanesulfonic acid, trifluoroacetic acid,
p-toluenesulfonic acid monohydrate, 10-camphorsulfonic acid,
concentrated sulfuric acid, dichloroacetic acid,
tetramethylammonium hydrogen sulfate and the like, and they can be
used alone or in combination, preferably, a mixture of
propylphosphonic anhydride and methanesulfonic acid. The condensing
agent may be used in an amount of 1 to 5 molar equivalents,
preferably 1 to 3 molar equivalents, to the compound (II').
[0073] The reaction temperature is not particularly limited, but
the reaction usually can be conducted at about 0 to 100.degree. C.,
preferably at 0.degree. C. to room temperature.
[0074] The reaction time is not limited, but the reaction usually
can be conducted for 0.5 hour to 24 hours, preferably for 1 to 10
hours.
Step 3
##STR00024##
[0075] wherein the variables are as defined above.
[0076] In this step, a compound of the formula (IV) is reacted with
a metal salt to obtain a compound of the formula (IV'').
[0077] The solvent is not limited so long as it allows the above
process to proceed efficiently. Examples of such solvent include
N-methylpyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide
and the like, which may be used alone or in combination.
N-methylpyrrolidone is preferable.
[0078] Examples of the metal salt include lithium chloride and
lithium bromide and the like, and lithium chloride is preferable.
The metal salt may be used in an amount of 1 to 20 molar
equivalents, preferably 5 to 10 molar equivalents, to the compound
(IV).
[0079] The reaction temperature is not particularly limited, but
the reaction usually can be conducted at about 0 to 100.degree. C.,
preferably at room temperature to 100.degree. C.
[0080] The reaction time is not limited, but the reaction usually
can be conducted for 0.5 hour to 48 hours, preferably for 12 to 24
hours.
Step 4
##STR00025##
[0081] wherein P.sup.r is a protecting group for a hydroxy group
such as an ester group or an ether group, and the other variables
are as defined above.
[0082] In this step, compound (V''') can be obtained according to a
conventional method for converting the hydroxyl group of compound
(IV'') to an ester group or an ether group. Examples for such
method can be found in Protective Groups in Organic Synthesis,
Theodora W Green (John Wiley & Sons), Prog. Med 5: 2157-2161
(1985) and Supplied by The British Library--"The world's
Knowledge".
[0083] As used herein, "diastereomer ratio" refers to the ratio of
the HPLC area percentage between the two stereoisomers shown
below.
##STR00026##
[0084] The compounds of formula (V) is useful for symptoms and/or
diseases induced by influenza viruses. It is useful for the
treatment, prevention, and/or symptom relief for example, cold
symptom involved with fever, chills, headache, muscle pain, and
feeling of generalized worthlessness, airway inflammation such as
sore throat, Nasal discharge, congested nose, cough, phlegm,
gastrointestinal symptom such as stomachache, emesis, diarrehea, in
addition, concomitant disease involved with secondary infection
such as acute encephalopathy, pneumonia.
[0085] The compound represented by the formula (VI) can be an
excellent medicine because it has advantages such as high oral
absorbability, good bioavailability, good clearance, high pulmonary
migration, and the like.
[0086] The compound represented by the formula (V) can be a
medicine with reduced side effects because it has high inhibitory
activity to cap-dependent endonuclease, which is a virus-specific
enzyme, and thus it has a highly specific effect.
[0087] Furthermore, the compound of the formula (V) and/or the
compound of the formula (VI) are excellent in terms of metabolic
stability, high solubility, high oral absorbability, good
bioavailability, good clearance, high lung transitivity, long
half-life, high binding rate to non-protein, low hERG channel
inhibition, low CYP inhibition, CPE (CytoPathic Effect) suppression
effect, and/or in that it also has advantages that it is negative
in phototoxicity test, Ames test, genotoxicity test, or has no
toxicity such as hepatic injury. Thus, the compound of the formula
(V) and/or the compound of the formula (VI) can be an excellent
medicament.
[0088] The compounds of the formula (V) and/or the compound of the
formula (VI) can be administered by oral or parenteral route. For
oral administration, the compounds of the formula (V) and/or the
compound of the formula (VI) can be used in any form of usual
formulations, for example, formulations in a solid form such as
tablets, powders, granules, capsules; formulations in a liquid form
such as aqueous formulation; oily suspension; syrup or elixir. For
parenteral administration, the compounds of the formula (V) and/or
the compound of the formula (VI) can be used in a form of aqueous
or oily suspending injection, or nose drops. In the preparation of
such formulation, conventional excipients, binding agents,
lubricants, aqueous solvents, oleaginous solvents, emulsifying
agents, suspending agents, preservatives, stabilizers, and the like
can be optionally used. A pharmaceutical composition comprising the
compound of the formula (V) and/or the compound of the formula (VI)
may be prepared by combining (for example, blending) a
therapeutically effective amount of the compound of the formula (V)
and/or the compound of the formula (VI) with a pharmaceutically
acceptable carrier or diluent.
[0089] For oral administration, daily dosage of the compound of the
formula (V) and/or the compound of the formula (VI) can be
approximately 0.05-3000 mg, preferably approximately 0.1-1000 mg
per day for an adult, while such dosage varies depending on the
administration route therefor, age, body weight, conditions of the
patient, and disease in the patient. The dosage may be divided for
administration, if necessary. In case of parenteral administration,
the daily dosage for an adult can be between approximately
0.01-1000 mg, preferably approximately 0.05-500 mg.
EXAMPLES
[0090] The present invention is explained in more detail with
reference to the Examples, Reference Examples, Preparation Examples
for intermediates, and Test Examples, but the present invention is
not limited to these examples.
[0091] The NMR analysis in the Reference Examples and Examples were
conducted using DMSO-d6, CDCl.sub.3 at 400 MHz.
Powder X-Ray Diffraction Pattern
[0092] Powder X-ray diffraction analysis of the crystal obtained in
each Example was conducted according to powder X-ray diffraction
analysis method in General Tests in Japanese Pharmacopoeia under
the following conditions.
(Device)
MinFlex600 RINT-TTRIII (Rigaku)
(Method)
[0093] Detector: High-speed one-dimensional detector (D/TecUltra 2)
and variable knife edge Measurement method: reflection method Type
of light source: Cu bulb Working wavelength: CuK.alpha. ray Tube
current: 10 mA, or 15 mA Tube voltage: 30 Kv, or 40 Kv Sample
plate: aluminum or glass X-ray incident angle (8): 3-40.degree.,
sampling width: 0.01.degree., or X-ray incident angle (8):
4-40.degree., sampling width: 0.02.degree.
[0094] In general, since diffraction angles (2.theta.) in powder
X-ray diffraction may involve errors within .+-.0.2.degree., the
values of the diffraction angle include values within the range of
about .+-.0.2.degree.. Therefore, the present invention includes
not only crystals in which the diffraction angles of peaks in
powder X-ray diffraction completely match but also crystals in
which diffraction angles of peaks coincide with errors of about
.+-.0.2.degree..
(Measurement of Water Content by Karl Fischer Method)
[0095] Water content was determined according to General Tests for
water determination (coulometric titration) in Japanese
Pharmacopoeia. Aquamicron.TM. AX (Mitsubishi Chemical Corporation)
was used as an anolyte solution, and Aquamicron.TM. CXU was used as
a catholyte solution.
[0096] In general, a water content measurement by Karl Fischer
Method may involve an error within the range of .+-.0.3%.
Accordingly, a specific value of water content as measured should
embrace any value within the range of .+-.0.3%.
TG/DTA Measurement
[0097] TG/DTA measurement of the crystals obtained in each example
was conducted. A sample was weighed in an aluminum pan and measured
in an open system. The measurement conditions are shown below.
Apparatus: TG/DTA 7200 (Hitachi High-Tech Science)
[0098] Measurement temperature range: 30.degree. C.-250.degree. C.
Heating rate: 10.degree. C./min
[0099] In general, TG/DTA measurement may involve an error within
the range of .+-.2.degree. C. Accordingly, a specific value as
measured should embrace any value within the range of .+-.2.degree.
C.
Dynamic Vapor Sorption (DVS)
[0100] Dynamic vapor sorption analysis of the crystals obtained in
each example was conducted. A sample was weighed in a sample pan
and measured under the conditions as follows.
Apparatus: DVS Advantage (Surface Measurement Systems Ltd.)
[0101] Measurement point: from 0% RH to 95% RH stepped 5%, then 95%
RH to 0% RH stepped 5%
Temperature: 25.degree. C. or 60.degree. C.
Measurement of Differential Scanning Calorimetry (DSC)
[0102] DSC measurement of the crystals obtained in each example was
conducted. A sample was weighed in a stainless steel pan with
hermetic seal and measured under the following conditions.
Apparatus: METTLER TOLEDO DSC 822e
[0103] Measurement temperature range: 30.degree. C.-300.degree. C.
Heating rate: 10.degree. C./min Atmosphere: N2 40 mL/min
[0104] In general, differential scanning calorimetry (DSC) may
involve an error within the range of .+-.2.degree. C. Accordingly,
a specific value as measured by differential scanning calorimetry
(DSC) should embrace any value within the range of .+-.2.degree.
C.
[0105] The meaning of each term in Examples is as follows.
DMA: N,N-dimethylacetamide
[0106] THF: tetrahydrofuran T3P: propylphosphonic anhydride (cyclic
trimer)
Example 1: Preparation of Compound 3
##STR00027##
[0107] Step 1: Compound 3
[0108] DMA (300 mL) was added to Compound 1 (100.00 g, 406 mmol)
and the mixture was stirred. Sodium hydrogencarbonate (44.41 g, 529
mmol), dimethyl sulfate (58.91 g, 467 mmol) and DMA (100 mL) were
added and stirred at 25.degree. C. for 7 hours. Synthetic
hydrochloric acid (16.90 g) and water (500 g) were added to the
reaction mixture, and the mixture was extracted twice with ethyl
acetate (1000 and 550 mL). The organic layer was washed with 5%
brine (300 g) and water (300 g). The combined organic layer was
concentrated to about 500 g under reduced pressure. Ethyl acetate
(350 mL) was added to the concentrate, and the resulting solution
was concentrated to about 500 g under reduced pressure. DMA (300
mL) was added to the concentrate, and the resulting solution was
concentrated to about 400 g under reduced pressure. Pyridinium
p-toluenesulfonate (265.42 g) and DMA (100 mL) were added to the
concentrate and the reaction mixture was heated to 60.degree. C. A
solution of tert-butyl carbazinate (69.80 g, 528 mmol) in DMA (100
mL) was added slowly to the reaction mixture over 6 hours. The
reaction mixture was stirred at 60.degree. C. for 3 hours and
cooled to 25.degree. C. Ethanol (100 mL) and water (290 mL) were
added to the reaction mixture, and the reaction mixture was warmed
to 30.degree. C. A mixture of ethanol (100 mL) and water (520 mL)
was added slowly to the reaction mixture. The reaction mixture was
cooled to 0.degree. C. and then stirred at 0.degree. C. for 1.5
hours. The resulting pale yellowish white precipitate was collected
by filtration. The resulting solid was washed with a mixture of
ethanol (480 mL) and water (720 mL), and dried to give monohydrate
of compound 3 (122.70 g, yield 77%) as a pale yellowish white
solid.
[0109] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta.:1.45 (s, 9H), 3.77
(s, 3H), 5.26 (s, 2H), 6.39 (d, J=7.6 Hz, 1H), 7.27-7.47 (m, 6H),
7.64-8.23 (br s, 1H)
[0110] Powder X-ray diffraction 2.theta. (.degree.): 5.4, 7.5, 8.4,
10.6, 11.9, 13.5, 20.2, 22.9
[0111] The powder X-ray diffraction pattern of Compound 3 is shown
in FIG. 1.
[0112] Water content by Karl Fischer method: 4.5%
Example 2: Preparation of Compound 9
##STR00028##
[0113] Step 1: Compound 6
[0114] Compound 5 (28.29 g, 167.4 mmol) and DMA (65 mL) were added
to compound 4 (20.00 g, 104.6 mmol), and the mixture was stirred.
After the mixture was warmed to 40.degree. C., sodium tert-butoxide
(15.09 g, 157.0 mmol) was added slowly. The reaction mixture was
stirred at 40.degree. C. for 3 hours and then cooled to 20.degree.
C. Acetic acid (3.14 g) and 10% sodium chloride aqueous solution
(64 g) were added to the reaction mixture, and the mixture was
extracted twice with ethyl acetate (60 mL). Water (144 mL) was
added to the combined organic layer and the mixture was cooled to
0.degree. C. The resulting pale yellowish white precipitate was
collected by filtration. The resulting solid was washed with a
mixture of methanol (5.4 g) and water (48.6 g), and dried to give
Compound 6 (20.44 g, yield 78%) as a pale yellowish white
solid.
[0115] .sup.1H-NMR (CDCl.sub.3) .delta.: 3.34 (s, 6H), 3.53 (d,
J=5.2 Hz, 2H), 3.76 (t, J=5.6 Hz, 2H), 3.90 (t, J=5.6 Hz, 2H), 4.43
(t, J=5.2 Hz, 1H), 7.70-7.73 (m, 2H), 7.84-7.87 (m, 2H)
Step 2 Compound 8
[0116] Ethanol (20 mL) and water (20 mL) were added to compound 6
(20.02 g, 71.68 mmol), and the mixture was stirred. The mixture was
warmed to 60.degree. C. The mixture was added with 60% hydrazine
monohydrate aqueous solution (8.99 g, 107.7 mmol) and stirred at
60.degree. C. for 4 hours. After addition of water (40 mL) followed
by cooling to 30.degree. C., 17% potassium hydroxide aqueous
solution (92.12 g) was added to the reaction mixture. The reaction
mixture was extracted four times with methylene chloride (120, 78,
78, 78 mL). The combined organic layer was washed with water (20
mL), and concentrated to about 160 g under reduced pressure. THF
(100 mL) was added to the concentrate, and the mixture was
concentrated to about 40 g under reduced pressure. THF (100 mL) was
added to the concentrate, and the mixture was concentrated to about
40 g under reduced pressure. THF (20 mL) was added to the
concentrate, and the mixture was concentrated to about 15 g under
reduced pressure to obtain 15 g of a solution of compound 7 in
THF.
[0117] The above THF solution of compound 7 (14.71 g), THF (7 g)
and 1,8-diazabicyclo[5.4.0]-7-undecene (379.0 mg) were added to
compound 3 (10.00 g, 25.5 mmol), and the mixture was stirred. The
reaction mixture was heated to 60.degree. C. and then stirred at
60.degree. C. for 24 hours. After the reaction mixture was cooled
to 25.degree. C., water (28 g) and acetic acid (3.72 g) were added.
The reaction mixture was extracted twice with ethyl acetate (50, 30
mL), and the organic layer was washed with 5% sodium
hydrogencarbonate aqueous solution (30 g) and water (28 g). The
organic layer was concentrated to about 36 g under reduced
pressure. Ethyl acetate was added to the reaction mixture, and the
resulting mixture was concentrated to about 36 g under reduced
pressure. Heptane (65 mL) was added to the concentrate and the
mixture was cooled to 5.degree. C. After stirring at 5.degree. C.
for 1 hour, the resulting pale yellowish white precipitate was
collected by filtration. The resulting solid was washed with a
mixture of heptane (32 mL) and ethyl acetate (14 mL), and dried to
obtain Compound 8 (10.10 g, yield 81%) as a pale yellowish white
solid.
[0118] .sup.1H-NMR (CDCl.sub.3) .delta.:1.44 (s, 9H), 3.32-3.48 (m,
12H), 4.41 (t, J=5.2 Hz, 1H), 5.29 (s, 2H), 6.38 (d, J=7.6 Hz, 1H),
7.11-7.50 (m, 7H), 8.46 (s, 1H).
Step 3: Compound 9
[0119] Acetonitrile (170 mL) and water (30 mL) were added to
compound 8 (19.99 g, 40.7 mmol), and the mixture was stirred. The
reaction mixture was heated to 60.degree. C., and methanesulfonic
acid (11.70 g, 121.7 mmol) was added slowly. The reaction mixture
was stirred at 60.degree. C. for 6 hours and then cooled to
25.degree. C. 30% sodium hydroxide aqueous solution (15.91 g) was
added to the reaction mixture, and the resulting mixture was
concentrated to about 100 g under reduced pressure. Water (50 mL)
was added to the concentrate, and the resulting mixture was
concentrated to about 100 g under reduced pressure. After stirring
the concentrate at 25.degree. C. for 30 minutes, the resulting
yellow precipitate was collected by filtration. The obtained solid
was washed with water (40 mL) and dried to obtain 0.5 hydrate of
Compound 9 (10.43 g, yield 76%) as yellow crystals.
[0120] .sup.1H NMR (400 MHz, DMSO-d.sub.6), .delta.: 2.95 (ddd,
J=13.7, 12.3, 4.3 Hz, 1H), 3.13 (dd, J=11.2, 10.0 Hz, 1H), 3.44
(td, J=11.9, 3.1 Hz, 1H), 3.96-4.08 (m, 2H), 4.14 (dd, J=13.9, 2.4
Hz, 1H), 4.80 (ddd, J=12.6, 9.9, 4.5 Hz, 1H), 5.08 (s, 2H), 6.22
(d, J=7.6 Hz, 1H), 7.24-7.41 (m, 4H), 7.52-7.60 (m, 2H), 7.69 (d,
J=7.6 Hz, 1H)
[0121] Powder X-ray diffraction 2.theta. (.degree.): 9.5, 13.4,
18.0, 19.3, 21.2, 22.5, 22.8, 23.6, 27.5, 28.1
[0122] The powder X-ray diffraction pattern of Compound 9 is shown
in FIG. 2.
[0123] Water content by Karl Fischer method: 2.8%
Example 3 Process of Compound 13
##STR00029##
[0124] Step 1: Compounds 11 and 12
[0125] Ethyl acetate (87 mL) and 50 (w/w) % T3P ethyl acetate
solution (145.80 g, 229.1 mmol) were added to 0.5 hydrate of
compound 9 (30.00 g, 89.2 mmol), and the mixture was stirred. The
reaction mixture was heated to 60.degree. C., triethylamine (18.55
g, 183.3 mmol) was added, and then
(R)-(+)-tetrahydrofuran-2-carboxylic acid (12.24 g, 105.4 mmol) was
added slowly. The reaction mixture was stirred at 60.degree. C. for
4.5 hours and then cooled to 0.degree. C., and the resulting pale
yellow precipitate was collected by filtration. The obtained solid
was washed with ethyl acetate (120 mL) to obtain Compound 11 (18.34
g, undried) as a pale yellow solid. Also, the filtrate and the
washing solution were combined to obtain an ethyl acetate solution
of Compound 12 (358.60 g).
Step 2: Compounds 13 and 9
[0126] Ethyl acetate (120 mL) and
1,8-diazabicyclo[5.4.0]-7-undecene (530 mg, 3.5 mmol) were added to
compound 11 (15.28 g), and the mixture was stirred. The reaction
mixture was heated to 30.degree. C., and a mixture of methanol
(1.67 g) and ethyl acetate (43 mL) was added slowly. The reaction
mixture was stirred at room temperature for 1 hour, and the
resulting white precipitate was collected by filtration. The
obtained crystals were washed with ethyl acetate (60 mL) and dried
to obtain white crystals of compound 13 (11.06 g, yield 45%).
[0127] .sup.1H-NMR (CDCl3) .delta.: 2.84-2.92 (m, 2H), 3.45 (td,
J=3.2 Hz, 12.0 Hz, 1H), 3.82 (dd, J=4.0 Hz, 11.2 Hz, 1H), 3.92 (dd,
J=4.4 Hz, 11.6 Hz, 1H), 4.13 (dd, J=2.8 Hz, 13.6 Hz, 1H), 4.47-4.54
(m, 1H), 4.96 (d, J=9.6 Hz, 1H), 5.27 (d, J=10.0 Hz, 1H), 5.76 (d,
J=13.2 Hz, 1H), 6.19 (d, J=7.6 Hz, 1H), 7.22 (d, J=8.0 Hz, 1H),
7.30-7.38 (m, 3H), 7.59 (dd, J=1.6 Hz, 8.0 Hz, 2H).
[0128] Powder X-ray diffraction 2.theta. (.degree.): 7.1, 14.1,
15.1, 21.0, 21.2, 22.9, 23.4
[0129] The powder X-ray diffraction pattern of Compound 13 is shown
in FIG. 3.
[0130] A solution of compound 12 in ethyl acetate (334.69 g) was
concentrated to about 170 g under reduced pressure. The concentrate
solution was stirred at 25.degree. C. Acetonitrile (224 mL), water
(56 mL) and 24% aqueous sodium hydroxide solution (150 g) was added
slowly to the mixture, and then separated into the organic layer
and the aqueous layer. Water (14 mL) was added to the aqueous layer
and extracted twice with acetonitrile (168 mL). The combined
organic layer was concentrated to about 250 g under reduced
pressure. The concentrate was heated to 60.degree. C., and
1,8-diazabicyclo[5.4.0]-7-undecene (19.01 g, 124.9 mmol) was added.
The reaction mixture was stirred at 60.degree. C. for 3.5 hours and
then cooled to 40.degree. C. 5.8% aqueous hydrochloric acid (50.40
g) was added to the reaction mixture, and the resulting mixture was
cooled to 25.degree. C. to obtain a solution (314.96 g). A portion
of the solution (158.86 g) was concentrated to about 85 g under
reduced pressure. The concentrate was stirred at 20.degree. C. for
2 hours, and water (28 mL) was added. The reaction mixture was
concentrated to about 100 g under reduced pressure. After stirring
the concentrate at 20.degree. C. for 1 hour, the precipitated pale
yellowish white crystals were collected by filtration. The obtained
crystals were washed with water (42 mL) and dried to obtain
Compound 9 (5.93 g, yield 42%) as pale yellowish white
crystals.
Example 4: Compound 19
##STR00030##
[0131] Step 1: Compound 15
[0132] Diisopropylamine (7.69 g, 76.0 mmol) was added to THF (25
mL), and the mixture was stirred and cooled to -40.degree. C. After
addition of 1.6 mol/L n-butyllithium (43.5 mL, 69.6 mmol), the
resulting mixture was stirred at 0.degree. C. for 1 hour. The
mixture was cooled to -40.degree. C., and a solution of
3,4-difluorobenzoic acid (5.00 g, 31.6 mmol) in THF (25 mL) was
added slowly. The reaction mixture was stirred at -40.degree. C.
for 1 hour, and N,N-dimethylformamide (5.74 g, 78.5 mmol) was added
slowly. To the reaction mixture was added 6 mol/L aqueous
hydrochloric acid (34.25 mL), and the mixture was warmed to
25.degree. C. and separated into the organic layer and the aqueous
layer. The aqueous layer was extracted with ethyl acetate (15 mL).
The combined organic layers was washed with water (5 mL). After
concentration under reduced pressure, toluene was added to the
residue to obtain a toluene solution of compound 15.
Step 2: Compound 16
[0133] Toluene (17.8 mL), thiophenol (3.90 g, 35.4 mmol) and
D-camphorsulfonic acid (1.16 g, 5.0 mmol) were added to the above
solution of compound 15. The mixture was stirred and heated to
60.degree. C. The reaction mixture was stirred at 60.degree. C. for
4 hours and then cooled to 5.degree. C. 2 mol/L sodium hydroxide
solution (10 mL) was added to the reaction mixture, and the
resulting mixture was warmed to 25.degree. C. The reaction mixture
was extracted with toluene (10 mL), and the organic layer was
washed with 2 mol/L sodium hydroxide (5 mL) and water (10 mL).
After concentration of the organic layer under reduced pressure,
toluene was added to obtain a toluene solution of compound 16.
Step 3: Compound 17
[0134] A mixture of aluminum chloride (5.52 g, 41.4 mmol) and
toluene (25 mL) was stirred and cooled to 0.degree. C. A solution
of 1,1,3,3-tetramethyldisiloxane (5.56 g, 41.4 mmol) in toluene (10
mL) was added dropwise to the reaction mixture, and the mixture was
warmed to 25.degree. C. The above toluene solution of Compound 16
was added slowly to the reaction mixture, and the mixture was
stirred at 25.degree. C. for 2.5 hours. After addition of 15%
sulfuric acid aqueous solution (35 mL), the mixture was stirred and
then separated into the organic layer and the aqueous layer. The
organic layer was washed twice with water (20 mL). The solution was
concentrated to about 16 g under reduced pressure. Heptane (40 mL)
was added slowly to the concentrate and cooled to 0.degree. C. The
resulting white precipitate was collected by filtration. The
obtained solid was washed with heptane (20 mL) and then dried to
obtain Compound 17 (7.20 g, yield 81.3%) as a white solid.
[0135] .sup.1H-NMR (CDCl.sub.3) .delta.:4.61 (d, J=1.6 Hz, 2H),
7.09-7.15 (m, 1H), 7.23-7.27 (m, 3H), 7.34-7.37 (m, 2H), 7.84-7.88
(m, 1H)
Step 4: Compound 18
[0136] Polyphosphoric acid (425.0 g) was stirred and heated to
80.degree. C. Compound 17 (85.0 g) was added, and the mixture was
warmed to 120.degree. C. and stirred at 120.degree. C. for 3 hours.
The reaction mixture was cooled to 80.degree. C., and water (200
mL) was added slowly. The reaction mixture was cooled to 30.degree.
C., and water (850 mL) was added. The mixture was extracted with
ethyl acetate (850 mL). The organic layer was washed with water
(425 mL) and 10% sodium hydrogencarbonate aqueous solution (255
mL). The solvent was evaporated under reduced pressure, and heptane
(340 mL) was added to the obtained residue. The solvent was
evaporated under reduced pressure, and heptane (85 mL) was added to
the obtained residue. After stirring the reaction mixture at
30.degree. C. for 30 minutes, the resulting brown precipitate was
collected by filtration. The obtained solid was washed with heptane
(42 mL) and then dried to obtain Compound 18 (72.0 g, yield 91%) as
a brown solid.
[0137] .sup.1H-NMR (CDCl3) .delta.:4.14 (d, J=1.0 Hz, 2H),
7.09-7.18 (m, 1H), 7.27-7.33 (m, 1H), 7.34-7.45 (m, 3H), 8.19 (dd,
J=8.5 Hz, 1.4 Hz, 1H)
Step 5: Compound 19
[0138] Sodium borohydride (234.0 mg, 6.2 mmol) was suspended in
0.5% sodium hydroxide aqueous solution (1.8 mL) to prepare a sodium
borohydride suspension. 2-Propanol (20 mL) and water (2.25 mL) were
added to compound 18 (4.5 g, 17.2 mmol). The mixture was stirred
and heated to 40.degree. C. The above sodium borohydride suspension
was added slowly to the mixture. The reaction mixture was stirred
at 40.degree. C. for 1.5 hours and cooled to 25.degree. C. Water
(32 mL) was added to the reaction mixture, followed by addition of
a mixed solution of water (6.7 mL) and 62% sulfuric acid aqueous
solution (460 mg). The reaction mixture was cooled to 5.degree. C.,
and the resulting brown precipitate was collected by filtration.
The solid was washed with water (18 mL) and then dried to obtain
Compound 19 (4.4 g, yield 97%) as a brown solid.
[0139] .sup.1H-NMR (CDCl3) .delta.: 2.67 (d, J=3.8 Hz, 1H), 4.20
(dd, J=14.4, 1.4 Hz, 2H), 4.68 (dd, J=14.5, 1.3 Hz, 2H), 7.02 (dt,
J=9.7, 8.3 Hz, 1H), 7.12-7.21 (m, 4H), 7.44-7.49 (m, 1H)
Example 5: Compounds (V) and (VI)
##STR00031##
[0140] Step 1-1: Compound 20
[0141] 1-Hexanol (22.5 g, 220 mmol) and THF (24.6 g) were combined,
and the temperature of the mixture was adjusted to 20.degree. C. A
solution of isopropyl magnesium chloride in THF (2 mol/L, 7.2 g,
14.7 mmol) was added to the mixture to prepare a solution of
magnesium hexoxide.
[0142] 1-Hexanol (22.5 g, 220 mmol) was added to compound 13 (12.0
g, 36.7 mmol) with stirring, and the temperature of the mixture was
adjusted to 20.degree. C. The above magnesium hexoxide solution was
added to the resulting slurry of compound 13. The reaction mixture
was stirred at 20.degree. C. for 4 hours, and then an aqueous
solution of citric acid (3.1 g of citric acid monohydrate and 36 g
of water) was added. The mixture was extracted with THF (10.7 g),
and the organic layer was washed with water (24 g). The organic
layer was concentrated to about 55 g under reduced pressure. A
solution of p-toluenesulfonic acid in THF (7.0 g of
p-toluenesulfonic acid monohydrate and 42.8 g of THF) was added to
the resulting concentrate. The mixture was concentrated to about 61
g under reduced pressure. THF (42.7 g) was added to the
concentrate, and the resulting mixture was concentrated to about 61
g under reduced pressure. After heating the mixture to 50.degree.
C., methyl tert-butyl ether (133.0 g) was added. The resulting
mixture was cooled to 10.degree. C., and stirred at 10.degree. C.
for 1.5 hours. The resulting white precipitate was collected by
filtration. The obtained solid was washed with a mixture of methyl
tert-butyl ether (40.0 g) and ethyl acetate (16.0 g), and dried to
obtain the tosylate of compound 20 (15.8 g, yield 87.2%) as white
crystals.
[0143] .sup.1H-NMR (CDCl.sub.3) .delta.: 0.88 (t, J=7.2 Hz, 3H),
1.25-1.34 (m, 4H), 1.34-1.43 (m, 2H), 1.76-1.85 (m, 2H), 2.34 (s,
3H), 3.04 (ddd, J=13.6, 11.7, 4.3 Hz, 3H), 3.36 (dd, J=11.6, 10.0
Hz, 3H), 3.43 (ddd, J=13.6, 12.0, 4.4 Hz, 3H), 4.00 (dd, J=11.7,
4.3 Hz, 1H), 4.06-4.18 (m, 4H), 4.80 (br, s, 1H), 7.16 (d, J=7.8
Hz, 1H), 7.62 (d, J=7.8 Hz, 1H), 7.62 (d, J=7.1 Hz, 1H), 8.17 (d,
J=7.1 Hz, 1H), 8.40 (br, s, 1H).
[0144] Powder X-ray diffraction 2.theta. (.degree.): 5.9, 8.4,
11.6, 12.7, 13.1, 15.7
[0145] The powder X-ray diffraction pattern of Compound 20 is shown
in FIG. 4.
Step 1-2: Compound 20
[0146] A reaction was carried out as described in Step 1-1 using a
solution of cyclohexylmagnesium chloride in THF (16.2 wt %, 0.4 eq)
instead of the solution of isopropylmagnesium chloride in THF (0.4
eq), and the reaction mixture was analyzed by HPLC to determine the
formation rate of compound 20.
[0147] HPLC area percentage of compound 20: 90.9% (RT=11.0 min) The
other procedures were the same as described in Step 1-1.
(Measurement Condition)
[0148] (1) Column: X Select.TM. CSH C18 (3.5 .mu.m i.d.
4.6.times.100 mm) (Waters)
[0149] Flow rate: 1.0 mL/min; UV detection wavelength: 254 nm;
[0150] Mobile phase:
[0151] [A] 0.1% formic acid aqueous solution, [B] acetonitrile
Gradient Program: (Concentration of [B]) 15%-15% 5 min; 15%-60% 10
min; 60%-85% 2 min; 85%-85% 3 min.
Step 1-3: Compound 20
[0152] 1-Hexanol (27.5 g, 270 mmol) was added to compound 13 (4.91
g, 15.0 mmol), and the mixture was stirred. The temperature of the
mixture was adjusted to 0.degree. C. A solution of sodium
tert-pentoxide in THF (1.4 mol/L, 45.0 mmol) was added to the
resulting slurry. After stirring at 0.degree. C. for 2.5 hours, the
reaction mixture was analyzed by HPLC to determine the formation
rate of compound 20.
[0153] HPLC area percentage of compound 20: 93.3% (RT=9.5 min)
(Measurement Condition)
[0154] (1) Column: CHIRALPAK.TM. IB (5.0 .mu.m i.d. 4.6.times.250
mm) (DAICEL)
[0155] Flow rate: 1.0 mL/min; UV detection wavelength: 254 nm;
[0156] Mobile phase: [A] 0.1% formic acid, [B] acetonitrile
[0157] Gradient Program: maintained with 35% Solvent [B] for 5 min;
linear gradient with 35% to 85% Solvent [B] over 6 min; and
maintained with 85% Solvent [B] for 2 min.
[0158] As shown above, it was found that the reaction proceeded in
good yield when using a magnesium salt or a sodium salt. The
desired product was obtained in high yield, especially when using
isopropyl magnesium chloride.
Step 2: Mesylate of Compound 21
[0159] Compound 19 (8.0 g, 30.3 mmol), ethyl acetate (48.7 g) and
cyclohexane (14.1 g) were added to compound 20 (12.0 g, 24.3 mmol),
and the mixture was stirred at 25.degree. C. 50 (w/w) % T3P ethyl
acetate solution (20.91 g, 32.9 mmol) was added followed by
addition of methanesulfonic acid (3.5 g, 36.4 mmol). The mixture
was heated to 60.degree. C. and stirred for 24 hours. After cooling
to 25.degree. C., THF (32.0 g) and water (24.0 g) were added, and
then 24% sodium hydroxide aqueous solution (30.8 g) was added
slowly. After settling, the mixture was separated into the organic
layer and the aqueous layer. The organic layer was washed twice
with 7% sodium chloride aqueous solution (60.0 g). A solution of
methanesulfonic acid (2.80 g, 29.1 mmol) in cyclohexane (9.3 g) and
ethyl acetate (32.1 g) was added to the combined organic layer. The
mixture was stirred at 25.degree. C. for 2 hours, and the resulting
white precipitate was collected by filtration. The obtained solid
was washed with ethyl acetate (43.3 g) and then dried to obtain
mesylate of compound 21 (13.65 g, yield 84.6%) as white
crystals.
[0160] .sup.1H-NMR (DMSO-d.sub.6) .delta.: 0.90 (3H, t, J=6.0 Hz),
1.29-1.36 (4H, m), 1.39-1.49 (2H, m), 1.67-1.79 (2H, m), 2.38 (3H,
s), 2.94 (1H, br s), 3.30 (1H, td, J=11.6, 2.4 Hz), 3.51 (1H, t,
J=10.4 Hz), 3.66 (1H, dd, J=11.2, 2.8 Hz), 3.92-4.01 (2H, m), 4.07
(1H, d, J=14.3 Hz), 4.20 (1H, s), 4.42-4.52 (1H, m), 5.43 (1H, dd,
J=14.4, 2.1 Hz), 5.79-5.83 (2H, m), 6.81 (1H, td, J=7.6, 1.2 Hz),
6.96 (1H, dd, J=7.8, 1.0 Hz), 7.09 (1H, J=8.0, 1.6 Hz), 7.12-7.18
(1H, m), 7.32 (1H, d, J=7.7 Hz), 7.37-7.49 (2H, m)
[0161] Powder X-ray diffraction 2.theta. (.degree.): 7.1, 9.3,
12.6, 14.1, 17.7, 18.7, 19.2, 22.2, 25.4, 27.7, 28.5, 37.8
[0162] The powder X-ray diffraction pattern of Compound 21 is shown
in FIG. 5.
[0163] DSC: Onset 216.degree. C., Peak 219.degree. C.
Step 3: Compound (V)
[0164] Lithium chloride (8.6 g, 203.3 mmol) was added to a mixture
of N-methylpyrrolidone (52.4 g) and compound 21 (15.0 g, 22.6
mmol), and the resulting mixture was heated to 75.degree. C. The
mixture was stirred at 75.degree. C. for 20 hours and then cooled
to 40.degree. C. Acetonitrile (20.0 g) was added to the reaction
mixture, followed by addition of water (11.6 g). After cooling the
mixture to 30.degree. C. and stirring for 30 minutes, water (142.5
g) was added slowly. After stirring at 30.degree. C. for 1.5 hours,
the resulting white precipitate was collected by filtration. The
solid obtained was washed with 2-propanol (60.1 g) and then dried
to obtain Compound (V) (9.91 g, yield 90.7%) as white crystals.
[0165] .sup.1H-NMR (CDCl3) .delta.: 3.00 (td, J=11.8, 3.2 Hz, 1H),
3.46 (td, J=12.0, 2.8 Hz, 1H), 3.59 (t, J=10.0 Hz, 1H), 3.82 (dd,
J=12.2, 3.0 Hz, 1H), 3.96 (dd, J=11.0, 3.0 Hz, 1H), 4.07 (d, J=13.6
Hz, 1H), 4.58 (dd, J=10.0, 2.8 Hz, 1H), 4.67 (dd, J=13.6, 2.0 Hz,
1H), 5.26-5.30 (m, 2H), 5.75 (d, J=8.0 Hz, 1H), 6.69 (d, J=7.6 Hz,
1H), 6.83-6.87 (m, 1H), 6.99-7.04 (m, 2H), 7.07-7.15 (m, 3H).
[0166] Powder X-ray diffraction 2.theta. (.degree.): 9.6, 10.9,
17.8, 21.5, 22.1, 23.5, 24.8
[0167] The powder X-ray diffraction pattern of Compound (V) is
shown in FIG. 6.
Step 4: Compound (VI)
[0168] Chloromethyl methyl carbonate (0.483 g, 3.10 mmol),
potassium carbonate (0.572 g, 4.14 mmol) and potassium iodide
(0.343 g, 2.07 mmol) were mixed with a suspension of Compound (V)
(1.00 g, 2.07 mmol) in DMA (5 ml). The mixture was heated to
50.degree. C. and stirred for 6 hours. DMA (1 ml) was added to the
reaction mixture, and the resulting mixture was stirred for 6
hours. The reaction mixture was cooled to room temperature, and DMA
(6 ml) was added. The mixture was stirred at 50.degree. C. for 5
minutes and then filtered. 1 mol/L hydrochloric acid (10 ml) and
water (4 ml) were added dropwise to the obtained filtrate under ice
cooling, and then, the mixture was stirred for 1 hour. The
precipitated solid was collected by filtration and dried under
reduced pressure at 60.degree. C. for 3 hours to obtain Compound
(VI) (1.10 g, 1.93 mmol, yield 93%).
[0169] .sup.1H-NMR (DMSO-D6) .delta.: 2.91-2.98 (1H, m), 3.24-3.31
(1H, m), 3.44 (1H, t, J=10.4 Hz), 3.69 (1H, dd, J=11.5, 2.8 Hz),
3.73 (3H, s), 4.00 (1H, dd, J=10.8, 2.9 Hz), 4.06 (1H, d, J=14.3
Hz), 4.40 (1H, d, J=11.8 Hz), 4.45 (1H, dd, J=9.9, 2.9 Hz), 5.42
(1H, dd, J=14.4, 1.8 Hz), 5.67 (1H, d, J=6.5 Hz), 5.72-5.75 (3H,
m), 6.83-6.87 (1H, m), 7.01 (1H, d, J=6.9 Hz), 7.09 (1H, dd, J=8.0,
1.1 Hz), 7.14-7.18 (1H, m), 7.23 (1H, d, J=7.8 Hz), 7.37-7.44 (2H,
m).
[0170] .sup.1H-NMR (DMSO-D.sub.6) .delta.: 2.91-2.98 (1H, m),
3.24-3.31 (1H, m), 3.44 (1H, t, J=10.4 Hz), 3.69 (1H, dd, J=11.5,
2.8 Hz), 3.73 (3H, s), 4.00 (1H, dd, J=10.8, 2.9 Hz), 4.06 (1H, d,
J=14.3 Hz), 4.40 (1H, d, J=11.8 Hz), 4.45 (1H, dd, J=9.9, 2.9 Hz),
5.42 (1H, dd, J=14.4, 1.8 Hz), 5.67 (1H, d, J=6.5 Hz), 5.72-5.75
(3H, m), 6.83-6.87 (1H, m), 7.01 (1H, d, J=6.9 Hz), 7.09 (1H, dd,
J=8.0, 1.1 Hz), 7.14-7.18 (1H, m), 7.23 (1H, d, J=7.8 Hz),
7.37-7.44 (2H, m).
Example 6: Preparation of Compounds 33 to 41 and Diastereomeric
Ratio of them
TABLE-US-00001 [0171] [Chem. 28] ##STR00032## Compound Compound
Yield diastereomer ratio (II) (IV) R.sup.2 % a:b 24 33 ##STR00033##
-- 3:1 25 34 ##STR00034## 50 6:1 26 35 ##STR00035## -- 3:1 27 36
##STR00036## 87 3.8:1 20 21 ##STR00037## 89 15.5:1 28 37
##STR00038## 24 2.3:1 29 38 ##STR00039## -- 1.9:1 30 39
##STR00040## -- 3.1:1 31 40 ##STR00041## 63 4.9:1 32 41
##STR00042## 89 6.3:1
Step 1: Compounds 24 to 32
[0172] Compounds 24 to 32 were prepared according to Steps 1-1,
1-2, and 1-3 of Example 5, as well as conventional methods.
Step 2: Compounds 33 to 41
[0173] With procedure of Step 2 described in Example 5, each of
Compounds 24 to 32 was reacted with Compound 19, and the reaction
mixture was analyzed by HPLC to determine the diastereomer ratio of
Compounds 33 to 41.
Compound 33a: tR 6.4 min/Compound 33b: tR 6.7 min Compound 34a: tR
8.9 min/Compound 34b: tR 9.3 min Compound 35a: tR 9.8 min/Compound
35b: tR 10.1 min Compound 36a: tR 10.7 min/Compound 36b: tR 11.1
min Compound 37a: tR 12.5 min/Compound 37b: tR 12.8 min Compound
38a: tR 13.4 min/Compound 38b: tR 13.8 min Compound 39a: tR 8.7
min/Compound 39b: tR 9.0 min Compound 40a: tR 9.9 min/Compound 40b:
tR 10.2 min Compound 41a: tR 10.6 min/Compound 41b: tR 11.0 min
(tR: retention time in HPLC measurement)
(Measurement Condition)
[0174] Column: KINETEX.TM. (2.6 .mu.m C18 i.d. 4.6.times.100 mm)
(Shimadzu)
[0175] Flow rate: 1.0 mL/min; UV detection wavelength: 254 nm;
[0176] Mobile phase: [A] 0.1% formic acid aqueous solution, [B]
0.1% formic acid in acetonitrile
[0177] Gradient Program: started with 25% Solvent [B]; linear
gradient with 25% to 70% Solvent [B] over 10 min; and maintained
with 70% Solvent [B] for 8 min.
Test Example 1: Measurement of Cap-Dependent Endonuclease (CEN)
Inhibitory Activity
1) Preparation of Substrate
[0178] 30merRNA (5'-pp-[m2'-O]GAA UAU(-Cy3) GCA UCA CUA GUA AGC UUU
GCU CUA-BHQ2-3', Japan Bioservice), wherein G at the 5' end has
been diphosphate-modified, the hydroxy group at 2' position has
been methoxylation-modified, U at the sixth position from the 5'
end has been labelled with Cy3, and the 3' end has been labelled
with BHQ2, was purchased, and a cap structure was added using
ScriptCap system manufactured by EPICENTRE to give the product m7G
[5']-ppp-[5'] [m2'-O]GAA UAU(-Cy3) GCA UCA CUA GUA AGC UUU GCU
CUA(-BHQ2)-3'). The product was isolated and purified by denatured
polyacrylamide gel electrophoresis, and used as a substrate.
2) Preparation of Enzyme
[0179] RNP was prepared from a virus particle according to standard
method (Reference: VIROLOGY (1976) 73, p 327-338 OLGA M.
ROCHOVANSKY). Specifically, 10 days old embryonated chicken egg was
inoculated with A/WSN/33 virus (1.times.10.sup.3 PFU/mL, 200
.mu.L). After incubation at 37.degree. C. for 2 days, the allantoic
fluid of the chicken egg was recovered. A virus particle was
purified by ultracentrifugation with 20% sucrose, solubilized with
TritonX-100 and lysolecithin, and an RNP fraction (50-70% glycerol
fraction) was collected by ultracentrifugation under density
gradient with 30-70% glycerol, and was used as an enzyme solution
(containing approximately 1 nM PB1/PB2/PA complex).
3) Enzymatic Reaction
[0180] 2.5 .mu.L of enzymatic reaction solution (53 mM
Tris-hydrochloride (pH 7.8), 1 mM MgCl.sub.2, 1.25 mM
dithiothreitol, 80 mM NaCl, 12.5% glycerol, 0.15 .mu.L of enzyme
solution) was dispensed into a 384-well polypropylene plate. Then,
0.5 .mu.L of a test compound solution which has been serially
diluted with dimethyl sulfoxide (DMSO) was added to the plate. For
positive control (PC) and negative control (NC), 0.5 .mu.L of DMSO
was added to the plate, respectively. The solutions were mixed
well. Then, 2 .mu.L of substrate solution (1.4 nM substrate RNA,
0.05% Tween 20) was added to initiate the reaction. After
incubation at room temperature for 60 minutes, 1 .mu.L of the
reaction solution was added to 10 .mu.L of Hi-Di formamide solution
(containing GeneScan 120 Liz Size Standard as a sizing marker:
manufactured by Applied Biosystem (ABI)) to quench the reaction.
For NC, the reaction was quenched by adding EDTA (4.5 mM) in
advance before the initiation of the reaction (the concentrations
as indicated are final concentration).
4) Measurement of Inhibition Rate (IC.sub.50 Value)
[0181] The reaction solution as quenched above was heated at
85.degree. C. for 5 minutes and then rapidly cooled on ice for 2
minutes, and analyzed on ABI PRIZM 3730 Genetic Analyzer. The peak
of the cap-dependent endonuclease product was quantified by the
analysis software ABI Genemapper. The CEN reaction inhibition ratio
(%) of the test compound was determined, with the fluorescence
intensity of PC and NC being 0% inhibition and 100% inhibition,
respectively, and the IC.sub.50 values were determined using a
curve fitting software (XLfit 2.0: Model 205 (IDBS)).
Test Example 2: CPE Suppression Effect
<Materials>
[0182] 2% FCS E-MEM (prepared by adding kanamycin and FCS to MEM
(Minimum Essential Medium) (Invitrogen))
[0183] 0.5% BSA E-MEM (prepared by adding kanamycin and BSA to MEM
(Minimum Essential Medium) (Invitrogen))
[0184] HBSS (hanks' Balanced Salt Solution)
[0185] MDBK cells (adjusted to appropriate cell number
(3.times.10.sup.5/mL) with 2% FCS E-MEM)
[0186] MDCK cells (prepared by washing twice with HBSS and adjusted
to appropriate cell number (5.times.10.sup.5/mL) with 0.5% BSA
E-MEM)
[0187] Trypsin solution (Trypsin from porcine pancreas (SIGMA) was
dissolved in PBS (-) and filtrated through a 0.45 .mu.m filter)
[0188] EnVision (PerkinElmer)
[0189] WST-8 Kit (Kishida Chemical Co., Ltd.)
[0190] 10% SDS solution
<Methods>
Diluting and Dispensing of Test Sample
[0191] As a culture medium, 2% FCS E-MEM was used for MDBK cells,
and 0.5% BSA E-MEM was used for MDCK cells. Same culture medium was
used for dilution of virus, cells and test samples.
[0192] The test sample was preliminarily diluted with a culture
medium to an appropriate concentration, and a 2- to 5-fold serial
dilution series was prepared (50 .mu.L/well) in a 96-well plate.
Two sets of the plate were prepared for anti-Flu activity
measurement and cytotoxicity measurement, respectively. The
measurements were performed in triplet for each drug.
[0193] When using MDCK cells for measurement of anti-Flu activity,
Trypsin was added to the cells so that the final concentration was
3 ug/mL.
Diluting and Dispensing of Influenza Virus
[0194] Influenza viruses were diluted to an appropriate
concentration with a culture medium and dispensed at 50 .mu.L/well
into 96-well plate containing the test sample. To the plate for
measuring cytotoxicity, 50 .mu.L/well of the culture solution was
dispensed.
Dilution and Dispensing of Cells
[0195] The cells were diluted to appropriate cell number and
dispensed at 100 .mu.L/well to a 96-well plate containing test
sample.
[0196] The cell culture was mixed using a plate mixer and incubated
in a CO.sub.2 incubator. The cells were cultured for 3 days for
anti-Flu activity measurement and cytotoxicity measurement.
Dispensing of WST-8
[0197] A 96-well plate cultured for 3 days was observed with naked
eye and under microscope to check the morphology of the cells and
the presence or absence of crystals. The supernatant was removed so
as not to inhale the cells from the plate.
[0198] A WST-8 Kit was diluted 10-fold with culture medium, and 100
.mu.L of WST-8 solution was dispensed into each well. After mixing
using a plate mixer, the cells were cultured in a CO.sub.2
incubator for 1 to 3 hours.
[0199] For measuring anti-Flu activity, after the plate was
incubated, 10% SDS solution (10 .mu.L) was dispensed into each well
to inactivate the virus.
Measurement of Absorbance
[0200] After mixing at 96-well plate, the absorbance was measured
on EnVision at two wavelengths of 450 nm/620 nm.
<Calculation of Measurement Values>
[0201] The values were calculated using Microsoft Excel or a
program having the equivalent calculation and processing ability,
based on the following equation.
[0202] Calculation of effective concentration to achieve 50%
influenza-infected cell death inhibition (EC.sub.50)
EC.sub.50=10.sup.Z
Z=(50%-High %)/(High %-Low %).times.{log(High conc.)-log(Low
conc.)}+log(High conc.)
[0203] The results of Test Example 1 and Test Example 2 for
Compound (V) are shown below.
Test example 1 (CEN IC 50): 1.93 nM, Test example 2 (CPE EC 50):
1.13 nM
[0204] The above results revealed that the compound of the formula
(V) shows high cap-dependent endonuclease (CEN) inhibitory activity
and/or high CPE inhibitory effect, and therefore, is useful as a
medicament for the treatment and/or prevention of symptoms and/or
diseases induced by infection with influenza virus.
[0205] Biological test examples of Compounds (V) and (VI) are
described below.
Test Example 3: CYP Inhibition Test
[0206] Using commercially available pooled human hepatic microsome
and employing, as a reference, typical substrate metabolism
reactions of human main five CYP enzyme forms (CYP1A2, 2C9, 2C19,
2D6, 3A4), i.e., 7-ethoxyresorufin O-deethylation (CYP1A2),
tolbutamide methyl-hydroxylation (CYP2C9), mephenytoin
4'-hydroxylation (CYP2C19), dextromethorphan O-demethylation
(CYP2D6), and terfenedine hydroxylation (CYP3A4), the degree of
inhibition by Compound (V) was assessed for each metabolite
production.
The reaction conditions were as follows:
[0207] Substrate: 0.5 .mu.mol/L ethoxyresorufin (CYP1A2), 100
.mu.mol/L tolbutamide (CYP2C9), 50 .mu.mol/L S-mephenytoin
(CYP2C19), 5 .mu.mol/L dextromethorphan (CYP2D6), 1 .mu.mol/L
terfenedine (CYP3A4); Reaction time: 15 minutes; Reaction
temperature: 37.degree. C.; Enzyme: pooled human hepatic microsome
0.2 mg protein/mL; Concentration of Compound (V): 1, 5, 10, 20
.mu.mol/L (four points).
[0208] For each of the five substances, a reaction solution was
prepared on a 96-well plate by adding the substrate, human hepatic
microsome and Compound (V) to 50 mmol/L Hepes buffer in the
proportion as described above. NADPH, which is a cofactor, was
added to initiate the metabolism reaction. After incubating at
37.degree. C. for 15 minutes, a methanol/acetonitrile solution (1/1
(v/v)) was added to quench the reaction. After centrifugation at
3000 rpm for minutes, resorufin (CYP1A2 metabolite) in the
supernatant was measured by fluorescent multi-label counter, and
hydroxytolbutamide (CYP2C9 metabolite), 4'-hydroxymephenytoin
(CYP2C19 metabolite), dextrorphan (CYP2D6 metabolite) and
terfenadine alcohol (CYP3A4 metabolite) were measured by
LC/MS/MS.
[0209] As a control, DMSO (the solvent for dissolving Compound (V))
was added solely to the reaction system. The remaining activity (%)
of Compound (V), relative to the control (100%), was calculated at
each concentration of the compound, and the IC.sub.50 was
calculated by reverse presumption by a logistic model using the
concentration and the inhibition rate.
(Result)
[0210] Compound (V): >20 .mu.mol/L for the five enzyme forms
Test Example 4: BA Test
[0211] Materials and methods for studies on oral absorption
(1) Animal: mouse or SD rat (2) Breeding condition: mouse or SD rat
was allowed to freely take solid feed and sterilized tap water. (3)
Dose and grouping: orally or intravenously administered at a
predetermined dose; grouping is as follows (Dose depends on the
compound) Oral administration: 1 to 30 mg/kg (n=2 to 3) Intravenous
administration: 0.5 to 10 mg/kg (n=2 to 3) (4) Preparation of
dosing solution: a solution or a suspension state for oral
administration; a solubilized state for intravenous administration
(5) Administration method: forced gastric administration using oral
probe for oral administration; administration from caudal vein with
a needle-equipped syringe for intravenous administration (6) End
point: blood was collected over time, and the plasma concentration
of Compounds (V) and (VI) was measured by LC/MS/MS. (7) Statistical
analysis: regarding the transition of the plasma concentration of
Compounds (V) and (VI), the area under the plasma
concentration-time curve (AUC) was calculated by non-linear least
squares program WinNonlin.TM., and the bioavailability (BA) of
Compounds (V) and (VI) was calculated from the AUCs of the oral
administration group and intravenous administration group.
(Result)
Compound (V): 4.2%
Compound (VI): 14.9%
[0212] The above results revealed that the prodrug has improved
bioavailability over the parent compound.
[0213] Accordingly, the compound of the formula (VI) is excellent
in oral absorption and is useful as a medicament in the treatment
and/or prevention of symptoms and/or diseases induced by infection
with influenza virus.
Test Example 5: Metabolism Stability Test
[0214] Compound (V) was reacted with commercially available pooled
human hepatic microsomes for a certain time. The remaining rate of
the compound was calculated by comparing the reacted sample and the
unreacted sample to assess the degree of metabolism of Compound (V)
in liver.
[0215] The compound was reacted in 0.2 mL of buffer (50 mmol/L
Tris-HCl pH 7.4, 150 mmol/L potassium chloride, 10 mmol/L magnesium
chloride) containing 0.5 mg protein/mL of human liver microsomes at
37.degree. C. for 0 minute or 30 minutes in the presence of 1
mmol/L NADPH (oxidative reaction). After the reaction, 50 .mu.L of
the reaction solution was added to 100 .mu.L of
methanol/acetonitrile=1/1 (v/v), mixed and centrifuged at 3000 rpm
for 15 minutes. The amount of Compound (V) in the supernatant was
measured by LC/MS/MS, and the remaining rate of the compound after
the reaction was calculated, with the amount of the compound at 0
minute of reaction time being 100%. The hydrolysis reaction was
carried out in the absence of NADPH, and the glucuronidation
reaction was carried out in the presence of 5 mmol/L UDP-glucuronic
acid instead of NADPH, and the subsequent procedure was carried out
in the same manner as described.
(Results)
[0216] The remaining rate in oxidative metabolism at 2 .mu.mol/L of
the compound is shown below.
Compound (V): 90.1%
Test Example 6: CYP3A4 Fluorescent MBI Test
[0217] The CYP3A4 fluorescent MBI test investigates enhancement of
CYP3A4 inhibition by Compound (V) in metabolism reaction.
7-benzyloxytrifluoromethylcoumarin (7-BFC) was debenzylated by
CYP3A4 enzyme (enzyme expressed in Escherichia coli) and a
metabolite, 7-hydroxytrifluoromethylcoumarin (7-HFC) which emits
fluorescent light was produced. The test was performed using 7-HFC
production reaction as an index.
[0218] The reaction conditions were as follows: Substrate, 5.6
.mu.mol/L 7-BFC; pre-reaction time, 0 or 30 minutes; reaction time,
15 minutes; reaction temperature, 25.degree. C. (room temperature);
CYP3A4 content (expressed in Escherichia coli), 62.5 .mu.mol/mL at
pre-reaction, 6.25 pmol/mL at reaction (at 10-fold dilution);
concentration of Compound (V), 0.625, 1.25, 2.5, 5, 10, 20
.mu.mol/L (six points).
[0219] A pre-reaction solution containing the enzyme and Compound
(V) in K-Pi buffer (pH 7.4) as described above was added to a
96-well plate. A part of the solution was transferred to another
96-well plate and 1/10 diluted with a substrate and K-Pi buffer.
NADPH, as a co-factor, was added to initiate the reaction (without
pre-incubation), and acetonitrile/0.5 mol/L Tris
(trishydroxyaminomethane)=4/1 (V/V) was added to quench the
reaction after incubation for a predetermined time. Also, to
another pre-incubation solution was added NADPH to initiate
pre-incubation (with pre-incubation). After pre-incubation for a
predetermined time, a part of the solution was transferred to
another plate and 1/10 diluted with a substrate and K-Pi buffer to
initiate reaction. After the reaction for a predetermined time,
acetonitrile/0.5 mol/L Tris (trishydroxyaminomethane)=4/1 (V/V) was
added to quench the reaction. For each of the plates on which the
reaction was performed, the fluorescent value of the metabolite
7-HFC was measured by fluorescent plate reader (Ex=420 nm, Em=535
nm).
[0220] As a control for remaining activity, DMSO (i.e., the solvent
for dissolving Compound (V)) was added solely to the reaction
system, and the remaining activity (%) was calculated for each
concentration of Compound (V) in the solution. The IC.sub.50 value
was calculated by reverse-presumption by logistic model using the
concentration and the inhibition rate. A difference of 5 .mu.M or
more in the IC.sub.50 values was defined as (+) and a difference of
3 .mu.M or less was defined as (-)
(Results)
Compound (V): (-)
Test Example 7: Fluctuation Ames Test
[0221] The mutagenicity of Compound (V) was evaluated.
[0222] Each 20 .mu.L of freeze-stored Salmonella typhimurium (TA98
and TA100 strain) was inoculated in 10 mL of liquid nutrient medium
(2.5% Oxoid nutrient broth No. 2), and the cultures are incubated
at 37.degree. C. under shaking for 10 hours. The TA98 culture (9
mL) was centrifuged (2000.times.g, 10 minutes) to remove medium,
and the bacteria was suspended in 9 mL of Micro F buffer
(K.sub.2HPO.sub.4: 3.5 g/L, KH.sub.2PO.sub.4: 1 g/L,
(NH.sub.4).sub.2SO.sub.4: 1 g/L, trisodium citrate dihydrate: 0.25
g/L, MgSO.sub.4.7H.sub.2O: 0.1 g/L). The suspension was added to
110 mL of Exposure medium (Micro F buffer containing Biotin: 8
.mu.g/mL, histidine: 0.2 .mu.g/mL, glucose: 8 mg/mL). The TA100
culture (3.16 mL) was added to 120 mL of Exposure medium to prepare
the test bacterial solution. The test bacterial solution (588
.mu.L), or in the case with metabolic activation system, a mixed
solution of the test bacterial solution (498 .mu.l) and the S9 mix
(90 .mu.L) was mixed with each 12 .mu.L of the following solutions:
Compound (V) in DMSO, serially diluted 2- or 3-fold in several
steps from maximum dose 50 mg/mL; DMSO as negative control; 50
.mu.g/mL of 4-nitroquinoline-1-oxide in DMSO as positive control
for TA98 without metabolic activation system; 0.25 .mu.g/mL of
2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide in DMSO as positive
control for TA100 without metabolic activation system; 40 .mu.g/mL
of 2-aminoanthracene in DMSO as positive control for TA98 with
metabolic activation system; or 20 .mu.g/mL of 2-aminoanthracene in
DMSO as positive control for TA100 with metabolic activation
system. The mixture was incubated at 37.degree. C. under shaking
for 90 minutes. The bacterial solution thus exposed to Compound (V)
(460 .mu.L) was added to 2300 .mu.L of Indicator medium (Micro F
buffer containing biotin: 8 .mu.g/mL, histidine: 0.2 .mu.g/mL,
glucose: 8 mg/mL, Bromo Cresol Purple: 37.5 .mu.g/mL), and each 50
.mu.L of the mixture was dispensed into a microplate (48 wells per
dose). After stationary cultivation at 37.degree. C. for 3 days, a
well containing bacteria, which has acquired a proliferative
ability by mutation in the gene encoding amino acid (histidine)
synthetase, turns the color from purple to yellow due to pH change.
The number of the yellow wells among the 48 wells per dose was
counted to evaluate the mutagenicity by comparing with the negative
control group. (-) means that mutagenicity is negative and (+)
means positive.
(Result)
Compound (V) (-)
Test Example 8: hERG Test
[0223] For the purpose of assessing risk of an electrocardiogram QT
interval prolongation, effects of Compound (V) on delayed rectifier
K+ current (IKr), which plays an important role in the ventricular
repolarization process, was investigated using HEK293 cells
expressing human ether-a-go-go related gene (hERG) channel.
[0224] Using an automated patch clamp system (PatchXpress 7000A,
Axon Instruments Inc.), a cell was maintained at a membrane
potential of -80 mV by whole cell patch clamp method. IKr induced
by depolarization pulse stimulation at +40 mV for 2 seconds, and
further, repolarization pulse stimulation at -50 mV for 2 seconds
were recorded. After the generated current was stabilized,
extracellular solution (NaCl: 135 mmol/L, KCl: 5.4 mmol/L,
NaH.sub.2PO.sub.4: 0.3 mmol/L, CaCl.sub.2.2H.sub.2O: 1.8 mmol/L,
MgCl.sub.2.6H.sub.2O: 1 mmol/L, glucose: 10 mmol/L, HEPES
(4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid): 10 mmol/L,
pH=7.4) containing Compound (V) at an objective concentration was
applied to the cell at room temperature for 10 minutes. From the
I.sub.Kr as recorded, absolute value of the tail peak current was
determined on the basis of the current value at the resting
membrane potential using an analysis software (DataXpress ver.1,
Molecular Devices Corporation). Further, the inhibition rate to the
tail peak current before applying Compound (V) was calculated, and
compared with the vehicle-applied group (0.1% dimethyl sulfoxide
solution) to assess influence of Compound (V) on I.sub.Kr.
(Result)
[0225] The inhibition rate at 0.3 to 10 .mu.mol/L of the compound
is shown below.
Compound (V): 7.9%
Test Example 9: Solubility Test
[0226] The solubility of Compound (V) was determined under 1% DMSO
addition condition. A 10 mmol/L solution of the compound was
prepared in DMSO, and 2 .mu.L of Compound (V) solution was added
respectively to 198 .mu.L of JP-1 solution (sodium chloride 2.0 g,
hydrochloric acid 7.0 mL and water to reach 1000 mL) and JP-2
solution (3.40 g of potassium dihydrogenphosphate and 3.55 g of
disodium hydrogenphosphate anhydrous dissolved in water to reach
1000 mL, followed by adding 1 volume of which to 1 volume of
water). After shaking at room temperature for 1 hour, the mixture
was filtered. The filtrate was ten-fold diluted with
methanol/water=1/1 (v/v), and the compound concentration in the
filtrate was measured using LC/MS by absolute calibration
method.
(Result)
[0227] Compound (V): 42.2 .mu.mol/L
Test Example 10: Powder Solubility Test
[0228] Appropriate amounts of Compound (V) were put into
appropriate containers. To the respective containers were added 200
.mu.L of JP-1 solution (sodium chloride 2.0 g, hydrochloric acid
7.0 mL and water to reach 1000 mL), 200 .mu.L of JP-2 solution (500
mL of water was added to 50 mL of phosphate buffer (pH 6.8)), and
200 .mu.L of 20 mmol/L sodium taurocholate (TCA)/JP-2 solution (TCA
1.08 g and water to reach 100 mL). If Compound (V) was fully
dissolved after addition to the test solution, Compound (V) was
added further as appropriate. The containers were sealed, and
shaken for 1 hour at 37.degree. C. The mixtures were filtered, and
100 .mu.L of methanol was added to each of the filtrate (100 .mu.L)
so that the filtrates were two-fold diluted. The dilution ratio was
changed if necessary. The dilutions were observed for bubbles and
precipitates, and then the containers were sealed and shaken.
Quantification of Compound (V) was performed by HPLC with an
absolute calibration method.
(Result)
[0229] Compound (V): JP-1 solution; 7.1 .mu.g/mL, JP-2 solution 4.4
.mu.g/mL, 20 mmol/L TCA/JP-2 solution 16.1 .mu.g/mL
Test Example 11 Ames Test
[0230] Ames test was performed using Salmonellas (Salmonella
typhimurium) TA 98, TA100, TA1535 and TA1537 and Escherichia coli
WP2uvrA as a test strain with or without metabolic activation in
the pre-incubation to check the presence or absence of gene
mutagenicity of Compound (V).
(Result)
Compound (V): (-)
Test Example 12: Photohemolysis Test
[0231] Compound (V) was dissolved at a predetermined concentration
and mixed on a microplate with a 0.1 to 0.0008% red blood cell
suspension (2.5 v/v %) prepared from ovine defibrinated blood of
sheep. Light irradiation in the UVA and UVB wavelength regions (10
J/cm.sup.2, 290-400 nm) was performed using ultraviolet fluorescent
lamp (GL20SE lamp, Sankyo Denki and FL20S-BLB lamp, Panasonic). The
mixed solution after the irradiation was collected and centrifuged.
After centrifugation, the supernatant was collected and transferred
to a microplate, and the absorbance (at 540 or 630 nm) of the
supernatant was measured. The absorbance at 540 and 630 nm were
used as an index of biological membrane damage (% light hemolysis)
and lipid membrane peroxidation (methemoglobin production),
respectively. (-): less than 10% for photohemolysis rate and less
than 0.05 for the change in absorbance at 630 nm; (+): 10% or more
for photohemolysis rate and 0.05 or more for the change in
absorbance at 630 nm.
(Result)
Compound (V): (-)
[0232] FIG. 7 and FIG. 8 show the time-course of the concentration
in plasma of Compound (V) and its prodrug Compound (VI) after oral
administration of Compound (VI) to rats under non-fasting
condition.
[0233] The concentration of Compound (VI) in plasma sample was
below the limit of quantification, indicating that Compound (VI)
which is a prodrug of Compound (V) was converted to Compound (V) in
vivo rapidly after the administration (see FIG. 8).
[0234] These test results reveal that a prodrug compound was
absorbed into the body after oral administration and rapidly
converted to its parent compound in blood. Therefore, Compounds (V)
and (VI) are useful as a medicament for the treatment and/or
prevention of symptoms and/or diseases induced by infection with
influenza virus.
Test Example 13: Intravenous Administration Test
Materials and Methods
[0235] (1) Test animal: SD rats (2) Rearing conditions: SD rats
allowed free access to solid feed and sterile tap water. (3) Dose
and grouping setting: Intravenously administered according to a
predetermined dosage. Groups were set up as follows (the dose may
be changed for each compound). Intravenous administration: 0.5 to 1
mg/kg (n=2 to 3) (4) Preparation of administration liquid:
Solubilized for intravenous administration. (5) Administration
method: From the tail vein with a needle-equipped syringe. (6) End
point: blood was collected over time, and the plasma concentration
of Compounds (V) was measured by LC/MS/MS (7) Statistical Analysis:
The total body clearance (CLtot) and elimination half-life (t1/2,
z) were calculated using the nonlinear least squares program
WinNonlin.TM. from the time-course of the concentration of Compound
(V) in plasma.
(Result)
Compound (V):
[0236] CLtot: 16.4 mL/min/kg t1/2, z: 3.4 hours
[0237] The above results revealed that Compound (V) has a low
whole-body clearance and a long half-life.
[0238] Accordingly, Compound (V) can be a drug that is excellent in
persistence and useful as a medicament for the treatment and/or
prevention of symptoms and/or diseases induced by infection with
influenza virus.
[0239] The compound and the process of the present invention are
useful as an intermediate for producing a useful compound as a
medicament for the treatment and/or prevention of symptoms and/or
diseases induced by infection with influenza virus. According to
the method of the present invention, the compound of the formula
(V) and the compound of the formula (VI) can be produced
efficiently.
* * * * *